Antibodies have proven to be effective therapeutic agents in humans for the treatment of a wide variety of disorders, including cancer, autoimmune diseases and infectious diseases. Although originally mouse monoclonal antibodies were tried as therapeutic agents, they generally proved to be unsuitable for use in humans due to the occurrence of a human anti-mouse antibody (HAMA) response. Rather, antibodies composed in part or entirely of human antibody amino acid sequences currently are the antibody agents of choice for use in humans. Of the numerous antibodies approved by the FDA for use in humans or currently in clinical trials, certain antibodies contain mouse variable regions linked to human constant regions and typically are referred to as chimeric antibodies. Others contain mouse CDRs within human framework and constant regions and typically are referred to as humanized antibodies. Still others are composed entirely of human-derived sequences (i.e., fully human variable and constant regions) and typically are referred to as human antibodies.
A number of approaches are known in the art for preparing human antibodies. In one type of approach, a library of human immunoglobulin sequences is screened on a display system (e.g., bacteriophage) with an antigen of interest to select antibody sequences having the desired antigenic specificity (see e.g., U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.). Since this approach is carried out in vitro, the human antibody sequences do not undergo affinity maturation or somatic mutation during the selection process, which may result in antibodies of lower affinity as compared to antibodies generated in vivo.
Thus, in another type of approach, mice whose genomes have been modified to contain human immunoglobulin sequences are used to raise antigen-specific antibodies by immunization with an antigen of interest. Such mice carry unrearranged human immunoglobulin genes (variable and constant regions) on transgenes and/or transchromosomes, which genes undergo apparently normal rearrangement and isotype switching in the mice. Moreover, somatic mutation occur during the maturation of the antibody response in these mice.
One example of such a mouse is the HuMAb Mouse® (Medarex, Inc.), which contains human immunoglobulin transgene miniloci that encode unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or κ and, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGκ monoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93 and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparation and use of HuMab mice, and the genomic modifications carried by such mice, are further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of all of which are hereby specifically incorporated by reference in their entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 by Korman et al.
An alternative transgenic mouse system for expressing human immunoglobulin genes is referred to as the Xenomouse (Abgenix, Inc.) and is described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al. Like the HuMAb Mouse® system, the Xenomouse system involves disruption of the endogenous mouse heavy and light chain genes and insertion into the genome of the mouse transgenes carrying unrearranged human heavy and light chain immunoglobulin loci that contain human variable and constant region sequences.
Other systems known in the art for expressing human immunoglobulin genes include the KM Mouse® system, described in detail in PCT Publication WO 02/43478 by Ishida et al., in which the mouse carries a human heavy chain transchromosome and a human light chain transgene, and the TC mouse system, described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727, in which the mouse carries both a human heavy chain transchromosome and a human light chain transchromosome. In each of these systems, the transgenes and/or transchromosomes carried by the mice comprise human immunoglobulin variable and constant region sequences.
U.S. Pat. No. 6,596,541 provides a prophetic example of a homologous recombinant mouse that expresses chimeric antibodies having human variable region sequences linked to mouse constant region sequences. In the example, the mouse heavy chain locus variable region (V-D-J segments) is precisely replaced with the human heavy chain V-D-J counterpart via a multi-step process. First, a large genomic fragment (greater than 20 kb) spanning the human immunoglobulin variable gene segments of interest is obtained and bacterial recombination is used to prepare a large targeting vector for use in eukaryotic cells (LTVEC) that includes homology arms totaling greater than 20 kb. The homology arms contain sequences from the endogenous mouse immunoglobulin locus. The LTVEC is then introduced into mouse embryonic stem cells. A cell in which homologous recombination has occurred between the LTVEC and the endogenous mouse immunoglobulin locus is identified using a quantitative assay to detect modification of allele (MOA) in the ES cells. No actual mice expressing chimeric antibodies, however, are described or characterized.